Most pathogenic bacteria elaborate large-molecular-weight (large-MW) surface polysaccharides, usually in the form of a capsule that coats and surrounds the bacterial surface. These surface carbohydrates offer substantial protection to bacterial pathogens against phagocytosis by migrating phagocytes and tissue-fixed macrophages—cells constituting a crucial host defense mechanism that limits microbial growth and spread and that probably accounts for much of the host's ability to avert microbe-induced diseases (Janeway et al., Immunobiology, 6th edition, Garland Science Publishing. New York, 2005). Specific polysaccharide structures function in different ways to affect bacterial interactions with the immune system and represent a paradigm for the way fine structures of carbohydrates can dictate the characteristics—and indeed the very nature—of immune responses.
Several decades ago, studies in mice showed that carbohydrates are T cell-independent antigens. Barrett, Adv. Pediatr., 32:139-158 (1985); Coutinho, Nature New Biol., 245:12-14 (1973); Coutinho et al., Eur J Immunol, 3:299-306 (1973); Guttormsen et al., Infect Immun, 67:6375-6384 (1999); McGhee et al., Microbial Immunol., 28:261-280 (1984). Purified polysaccharides induce specific IgM responses, with minimal IgG switching. A failure to induce immunoglobulin class switching from IgM to IgG isotypes and a lack of increased antibody production after rechallenge with antigen are hallmarks of a classic T cell-independent immune response. The conjugation of polysaccharides to proteins to allow carbohydrate-specific responses that elicit T-cell help has improved the efficacy of vaccines. See Guttormsen et al., supra; Sood et al., Expert Opin Investig Drugs, 7:333-347 (1988). The current generation of glycoconjugate vaccines has been one of the great success stories in the biomedical sciences: in most immunized populations, infections with Haemophilus influenzae type b, the vaccine types of Streptococcus pneumoniae in children, and Neisseria meningitidis (except group B) have been nearly eliminated. Lesinski and Westerink, Curr, Drug Targets Infect. Disord., 1:325-334 (2001); Weintraub, Carbohydr. Res., 338:2539-2547 (2003).
The current mechanistic paradigm is that protective responses to glycoconjugate vaccines are based on a “trick” played on the immune system. Guttormsen et al., supra; Lesinski and Westerink, J. Microbiol. Methods, 47:135-149 (2001). Presumably, a B cell that is specific for production of anti-polysaccharide antibodies takes up the glycoconjugate and presents a peptide from the covalently linked protein to T cells that recognize the peptide in the context of the MHC molecule. Stimulation of the B cell (with consequent production of carbohydrate-specific antibody) and activation of the peptide-recognizing CD4+ T cell result in T-cell help, which promotes immunoglobulin class switching to IgG and memory responses. Immunoglobulin class switching and B-cell memory depend on co-stimulation of the B cell through CD80 and/or CD86 interacting with CD28, through CD40 interacting with the CD40 ligand, and likely through interactions with other co-stimulatory molecules.
T cells are, however, able to recognize carbohydrates. There have been several reports on glycopeptide processing and MHC I/II presentation and recognition by T cells. Abdel et al., Eur J Immunol, 26:544-551 (1996); Corthay et al., Eur J Immunol, 28:2580-2590 (1998); Dong et al., Nature, 409(6816):97-101 (2001); Dzhambazov et al., Eur J Immunol, 35:357-366 (2005); Hanish and Ninkovic, Curr Protein and Peptide Sci, 7:307-315 (2006); Haurum et al., J Exp Med, 180:739-744 (1994); Hudrisier et al., J Biol Chem, 274:36274-36280 (1999); Jensen et al., J Immunol, 158:3769-3778 (1997); Michaelson et al., Eur J Immunol, 22:1819-1825 (1992); Vlad et al., J Exp Med, 196:1435-1446 (2002); Werdelin et al., Proc Natl Acad Sci USA, 99:9611-9613 (2002). Most of these studies have investigated the processing and presentation of post-translationally glycosylated proteins. These epitopes appear to be simple mono- or di-saccharides linked to peptides. It has been shown that MHCI binds to the glycopeptides—and not just the peptide portion—of these types of epitopes. In addition, T-cell receptor (TCR) binding to the processed glycopeptides has been shown to be dependent on contact of the TCR with the epitope formed by both the glycan and the peptide that is bound to MHC. Haurum et al., J Exp Med, 180:739-744 (1994). For instance, Holmdahl and colleagues showed that epitope glycosylation plays a critical role in T-cell recognition of type II collagen (CII). Corthay et al., Eur J Immunol, 28:2580-2590 (1998); Dzhambazov et al., Eur J Immunol, 35:357-366 (2005); Michaelson et al., Eur J Immunol, 22:1819-1825 (1992). These investigators have found that the immunodominant T-cell epitope in healthy joint cartilage of humans and rats is O-glycosylated. Studies of glycopeptide epitopes derived from tumor antigen mucin-like glycoprotein1 (MUC1) are of particular importance. Hanish and Ninkovic, supra. In tumor cells, protein glycosylation forms tumor-specific glycopeptide epitopes that can be recognized by CD4+ T cells. Vlad et al. (2002), supra; Werdelin et al., Proc Natl Acad Sci USA, 99:9611-9613 (2002). Changes in glycosylation patterns can alter epitope recognition by T cells. Hanish and Ninkovic, supra.
Over the past 15 years several successful vaccines in which polysaccharides are the key component have been introduced into clinical use. The most successful of these vaccines have been glycoconjugates in which the capsular polysaccharide (CPS) of a bacterial target is coupled to a protein carrier; this coupling induces T cell help to promote IgM-to-IgG switching, long-lived responses, and immunogenicity in children. The success of these vaccines varies with the population being immunized and with the characteristics of the specific vaccine. For example, pneumococcal conjugates have met with very good success in children, but have not lived up to expectations in adults (Siber et al., Science 265(5177):1385-1387 (1994).
The most widely accepted hypothesis underlying strategies for the preparation of glycoconjugate vaccines is that such vaccines are taken up through polysaccharide-specific B cells possibly after processing by dendritic cells and/or macrophages (Guttormsen et al., Inf. Imm. 67(12):6375-6384 (1999); Jones, Anais da Academia Brasileira de Ciências 77(2):293-324 (2005)). A peptide epitope of the carrier protein is subsequently presented to T cells. The resulting help provided to the polysaccharide-specific B cells by these activated T cells induces production of polysaccharide-specific IgG even though the T cells are activated by the MHC II bound peptides. However, little or no information exists on whether or not the T-cell epitopes are formed by the peptides generated from the glycoconjugate carriers or by epitopes formed by both the carbohydrate and peptides.